1. Field of the Invention
The present invention relates to a new enzyme, its method of production and its application to the preparation of methyl N-(L-aspartyl-1) L-phenylalaninate.
Enzymatic processes are currently being used to an ever increasing extent to obtain substances having a physiological activity. These enzymatic processes are convenient and, very often, they provide directly the required product with the desired configuration, without it being necessary to carry out separation of enantiomers or diastereoisomers.
2. Description of the Prior Art
Methyl N-(L-aspartyl-1) L-phenylalaninate, commonly known as aspartime, is a very well known dipeptide which possesses remarkable sweetening properties (R. H. Mazur et al., J. Amer. Chem. Soc., 1969, 91, 2684). It can be obtained directly by enzymatic processes starting from methyl DL-phenylalaninate and L-aspartic acid (European Patent Application Nos. 0 0154 472, 0 124 313 and 0 074 095); however, the productivity of these processes is low. This compound is also generally prepared either by using conventional chemical methods or by hydrogenating cutting of its derivative N-benzyloxy-carbonyl resulting from the enzymatic condensation of methyl DL-phenylalaninate with L-N-benzyloxy-carbonyl aspartic acid.
The Applicants have also developed a method of this type, using a proteolytic system, designated SP, extracted from a culture broth of Micrococcus caseolyticus, to obtain N-benzyloxy-carbonyl aspartame starting from L-N-benzyloxy-carbonyl aspartic acid and methyl DL-phenylalaninate.
Micrococcus caseolyticus, a micro-organism isolated from cow's milk and a strain of which was deposited at the Collection Nationale de Micro-Organisme at the Institut Pasteur in Paris, under No. 1194 on 28th Apr. 1982, does in fact provide, under certain culture conditions, a proteolytic system, SP, from which it is possible to extract a single neutral exocellular proteolytic protease, with a molecular weight of between 35,000 and 41,000, designed P in the following, exhibiting almost the complete proteolytic activity of the proteolytic system SP (M. Desmazeaud et al., Ann. Bio. Anim. Biochem. Biophys., 1968, 8, 419-429, 565-577 and European J. Biochem. 1971,, 19, 51-55).